Facioscapulohumeral muscular dystrophy (FSHD) is linked to a shortened 3.3 kb D4Z4 repeat array from 11-100 to 1-10 copies on the subtelomeric region of chromosome 4q35. The shortening of the D4Z4 array is believed to have a de-repression effect on genes in or near the D4Z4 region. In the parent R01, we proposed to test a hypothesized model of FSHD involving direct regulatory relationships between 4q35 deletions, double homeobox protein 4 (DUX4), and Paired-like homeodomain transcription factor 1 (PITX1). In aim 1, we proposed to determine whether Pitx1 was a direct transcriptional target of DUX4 by characterizing a putative DUX4 binding site in the promoter region of Pitx1 gene. In aim 2, we proposed to generate and characterize a conditional muscle-specific Pitx1 transgenic mouse model. In aim 3, we proposed to define molecular transcriptional pathways downstream of Pitx1 expression using the Pitx1 transgenic model. Supported by the parent R01, we were able to showed that DUX4 indeed is a transcriptional regulator of the PITX1 (Dixit et al., 2007). In addition, the Pitx1 transgenic mouse model generated in aim2 showed muscle atrophy phenotype with pathology similar to FSHD. In this revision, we propose to test the hypothesis that repressing the expression of genes involved in FSHD will rescue the pathology and phenotype of the disease. In the new aim 4, we proposed to use morpholino antisense oligos to repress the expression of DUX4, DUX4c and PITX1 using both cell culture and in vivo systems. The goal is to evaluate the feasibility of using morpholinos as a potential therapeutic mean for treating FSHD. In aim 4A, we will first identify a suitable cell model for testing the efficacy of the morpholinos. In aim 4B, we will determine the efficacy of the morpholinos and the effects of target gene suppressing in vivo by intramuscular injection of the morpholinos. In aim 4C, we will systematically deliver vivo-morpholinos against Pitx1 to the Pitx1 transgenic mice. The goal is to determine if the vivo-morpholinos can suppress the transgene and reverse the phenotype, as well as to determine the toxicity of systemic delivered vivo-morpholinos. The findings of the studies will provide important information of using anti-sense technology in treating FSHD. PUBLIC HEALTH RELEVANCE: Facioscapulohumeral muscular dystrophy (FSHD) is linked to a shortened 3.3 kb D4Z4 repeat array from 11-100 to 1-10 copies on the subtelomeric region of chromosome 4q35. The shortening of the D4Z4 array is believed to have a de-repression effect on genes in or near the D4Z4 region. Based on our preliminary data, we developed a pathophysiological model of FSHD involving direct regulatory relationships between 4q35 deletions, double homeobox protein 4 (DUX4), and paired-like homeodomain transcription factor 1 (PITX1). In the parent R01, we proposed to test the hypothesis that DUX4 is a transcription regulator of PITX1 and PITX1 activates genes involved in muscle atrophy in skeletal muscles. In aim 1, we proposed to determine if Pitx1 was a direct target of DUX4 by characterizing a putative DUX4 binding site in the promoter region of Pitx1 gene. We also proposed to identify additional downstream molecular targets of DUX4 in the aim. In aim 2, we proposed to generate and characterize a conditional muscle-specific Pitx1 transgenic mouse model and to test whether the induction of downstream genes involved in muscle wasting depends on the anatomical location of the muscle of the transgenic mice. In aim 3, we proposed to define molecular transcriptional pathways downstream of Pitx1 expression using the Pitx1 transgenic model and to determine whether the effect of Pitx1 over-expression is reversible. This is the third year of the grant and we have demonstrated that DUX4 is a transcriptional regulator of PITX1 (Dixit et al., 2007). In addition, we have generated and characterized the Pitx1 transgenic mouse model (manuscript in preparation). The Pitx1 transgenic animals showed muscle atrophy phenotype and pathology similar to FSHD (preliminary data). In this revision, we propose to test the hypothesis that repressing the expression of genes involved in FSHD will rescue the pathology and phenotype of the disease. In the new aim 4, we propose to use mopholino antisense oligos to repress the expression of DUX4, DUX4c and PITX1 using both cell culture and in vivo systems. The goal is to evaluate the feasibility of using morpholinos as a potential therapeutic mean for treating FSHD. In aim 4A, we will first identify a suitable cell model for testing the efficacy of the morpholinos and potentially other small molecules for developing therapeutic means. In aim 4B, we will determine the efficacy of the morpholinos and the effects of target gene suppressing in vivo by intramuscular injection of the morpholinos. In aim 4C, we will systematically deliver vivo-morpholinos against Pitx1 to the Pitx1 transgenic mice. The goal is to determine if the vivo-morpholinos can suppress the transgene and reverse the phenotype, as well as to determine the toxicity of systemic delivered vivo-morpholinos. The development of effective therapeutic means necessitates an in depth understanding of the cellular and molecular mechanisms mediating muscle atrophy in FSHD. The data generated by the support of the parent R01 supported that a regulatory pathway involving aberrant expression of DUX4, activation of PITX1 by DUX4 and PITX1 downstream genes involved in FSHD. In the new aim, we proposed to suppress the DUX4, DUX4c and PITX1 genes using morpholinos to identify molecules that can potentially used to suppress the translation of these genes in FSHD. Both cell cultures and in vivo systems will be used to determine the efficacy, toxicity, and feasibility of the approach. The studies will also provide insights on molecular functions of these genes and help develop and evaluate a potential cell model for drug screening for FSHD. In this proposal, the old aims and some preliminary data in the original proposal were not included to free up space for the new texts. All the other sections were updated with the additions marked in bold brackets. How the revision will accelerate the tempo of scientific research and allow for job creation and retention. The proposed study will help: 1. To develop and evaluate a cellular model for studying genes over-expressed in FSHD and drug screening. 2. To test the efficacy, toxicity and feasibility of using morpholinos to treat FSHD. 3. To increase understanding of functions of the FSHD candidate genes by suppressing their protein translation in cells and in muscles. To complete these aims, one graduate student (Ms. Vishakha Sharma) will be retained and one research associate will be hired. Ms. Sharma who performed the preliminary studies during her rotation will be able to stay in my laboratory to complete her project proposed in aim 4A and 4B. A research associate will be hired to assist the experiments proposed in the aims with focus on aim 4C.